Biophysical properties of cellular microenvironments can act to direct the self-renewal and differentiation fate outcomes of stem cells in multiple tissue types. As an example, the stem cells resident in skeletal muscles (MuSCs, also called satellite cells) are highly sensitive to elasticity of their surrounding ?niche? microenvironment. In culture models, elastic substrates corresponding to the more stiff microenvironments observed in muscle aging and diseases such as muscular dystrophy can induce MuSCs to differentiate instead of self-renew, which is a hallmark of muscle dysfunction in these pathological settings. Characterization of the viscoelastic properties of endogeneous stem cell niches within intact 3D tissues ex vivo or in vivo would provide critical insights into the consequence of microenvironmental mechanical changes on stem cell fate outcomes, but is not possible with current rheology and imaging approaches. Thus, new imaging methods are needed to achieve 3D mapping of local mechanical properties in and around individual anatomically-defined stem cell niches with cellular resolution and capable of non-destructive longitudinal assessment. The central objective of this proposal is to demonstrate photonic force optical coherence elastography (PF-OCE) for volumetric imaging of the mechanical properties of individual stem cell microenvironments in intact tissue explants and engineered biomaterial niches. PF-OCE will perform mechanical loading (`palpation') using photonic radiation pressure, combined with ultra-precise phase- sensitive optical coherence tomography (OCT), to detect the resulting displacements. Our approach differs from conventional laser tweezers-based active microrheology that uses optical gradient forces to generate transverse bead oscillations, and instead, PF-OCE utilizes low-NA on-axis radiation-pressure forces, which is a key factor enabling large-scale volumetric interrogation. We will apply PF-OCE to a muscle stem cell system, evaluating both biomimetic hydrogel culture substrates, previously developed by the Co-Investigator to mimic the viscoelastic properties of skeletal muscle tissue, and in muscle myofiber explant tissue, which maintains key molecular and mechanical aspects of the muscle stem cell niches in short-term culture. Specific Aim 1 will develop PF-OCE for quantitative reconstruction of viscoelastic properties of homogeneous muscle-mimetic poly(ethylene glycol) hydrogels, and demonstrate the ability to distinguish between photonic force (mechanical) and photothermal (heating) responses. Shear rheometry will be used to validate PF-OCE reconstructions of shear storage and loss moduli. Specific Aim 2 will apply PF-OCE to image the viscoelastic properties of individual muscle stem cell niches in intact muscle fiber explants and correlate them with stem cell fate outcomes. Stem cell fates, tracked via time-lapse two-photon microscopy, will be co-registered with 3D volumetric mechano-maps throughout myofiber explants from normal and dystropic mouse models. This new platform for volumetric mechano-microscopy could lead to a deeper understanding of spatiotemporal variations in stem cell niche mechanics, and establish new mechano-biomarkers as hallmarks of tissue dysfunction.